The major findings of this study are as follows: (i) glibenclamide attenuated LPS-induced myocardial injury in STZ-mice; (ii) glibenclamide reduced serum IL-1β and TNF-α induced by LPS in STZ-mice; (iii) glibenclamide inhibited Nalp3 and Caspase-1 expression by LPS + high glucose stimulation in cultured primary peritoneal macrophages. These together suggested that glibenclamide might protect against myocardial injury under inflammation in diabetes.
Sepsis is a common disease with a growing morbidity around the world. Severe sepsis and systemic inflammation are the leading causes of mortality in critically ill patients, resulting from a systemic oxidative-mediated inflammatory response to severe bacterial infection . It is well known that acute infections lead to difficulty in controlling blood glucose and that infection is the most frequently documented cause of ketoacidosis during diabetes mellitus . In this study, the endotoxemic mice displayed hypotension, decreased heart rate, and elevated serum epinephrine level after LPS stimulation, which was consistent with previous reports ,,. Prevention or management of sepsis remains a barrier to the successful care of many surgical and traumatic patients with diabetes, needing novel therapies urgently . Type 1 diabetes mellitus is a chronic, multifactorial autoimmune disease that involves the progressive destruction of pancreatic β-cells, ultimately resulting in the loss of insulin production and secretion . This response includes the production of cytokines such as IL-1β that orchestrate the recruitment of inflammatory cells to the islets and mediate direct cytotoxic effects on β-cells . TNF-α is considered to be a possible therapeutic target because it was up-regulated in multiple rodent-obesity models and TNF-α blunted insulin signaling in insulin targeting tissues. It is not only exacerbates inflammatory response through acting as a signal amplifier to induce other inflammatory cytokines production, but also contributes to myocardial hypertrophy and fibrosis, leading to left ventricular remodeling and dysfunction .
Experimental and clinical studies have suggested that diabetic state causes a specific diabetic cardiomyopathy independent of vascular complications. This cardiomyopathy is characterized by myocyte hypertrophy and fibrosis and may be responsible for the high incidence of cardiac dysfunction and mortality . Ventricles from diabetic patients show accumulation of glycoproteins, collagen, and active fibroblasts . Elevated inflammatory cytokines have been found in circulation and in the hearts of diabetic patients, contributing to heart failure. Cardiac overexpression of TNF-α has been associated with cardiac hypertrophy and fibrosis, as well with left ventricular dysfunction . Moreover, up-regulation of inflammatory cytokines and chemokines by subcutaneous injection of LPS was significantly more rapid and more pronounced in the diabetic mice compared with normal mice . These indicate that cardiac tissue is sensitive to inflammation and inflammatory response is stronger in diabetic condition. We observed significant elevations of IL-1β and TNF-α levels in serum and cardiac tissue upon LPS challenge in mice, suggesting that both of circulating and local inflammatory reaction may cause cardiac injury under diabetic and septic condition. In addition, we found that glibenclamide was able to inhibit the inflammatory cytokines secreted by peritoneal macrophages after LPS treatment. Interestingly, glibenclamide only inhibited the increases of IL-1β but not TNF-α in vitro. We considered this specific inhibition on IL-1β level by glibenclamide implicated the involvement of Nalp3, which regulates IL-1β but not TNF-α. There was an inconsistency of TNF-a data in serum and in the supernatant. We think that the situation in vivo is rather complex while the cell model in vitro is simple. The serum TNF-α levels might be influenced by many factors. Glibenclamide may lower the serum TNF- α level in a macrophage-independent manner.
KATP channels are a type of Kir constituted by heteromultimers of two kinds of proteins. Each channel is formed from four pore-forming Kir subunits (Kir 6.1 or Kir 6.2) complexes with four regulatory sulfonylurea receptor proteins (SUR1 in neuronal/pancreatic beta cells or SUR2 in cardiovascular cells). Glibenclamide is a sulfonylurea drug which binds to the SUR1 domain with 10 to 500 fold higher affinity than to the SUR2 domains . Previous study demonstrated that glibenclamide was able to suppress NALP3 activation independently of KATP. Thus, in this work, we used glibenclamide as an inhibitor of NALP3.
The mechanism of glibenclamide attenuated LPS-induced inflammation is not fully elucidated. Transient receptor potential melastatin 4 (TRPM4), a calcium-activated non-selective cation channel, is functionally expressed in the heart. TRPM4 has been linked to diverse physiological functions, such as protection against Ca2+ overload by cell membrane depolarization, modulation of Ca2+ oscillations controlling cytokine production in T lymphocytes and mast cells, and dendritic cell migration ,. Grand et al. reported that TRPM4 is inhibited by glibenclamide, a modulator of ATP binding cassette proteins (ABC transporters), such as the cystic fibrosis transmembrane conductance regulator (CFTR) . It was also reported that TRPM4 inhibitors 9-phenanthrol and glibenclamide could attenuate LPS-induced endothelial cell death and hypoxia and re-oxygenation-induced early after depolarizations . Glibenclamide is a potent blocker of the ATP-modulated K+ channel in insulin secreting cells and a broadly used anti-diabetic drug. Interestingly, recent study showed that glibenclamide could decrease TNF-α and NF-κB activation after subarachnoid hemorrhage . Moreover, glibenclamide reduced LPS-induced release of IL-1β、TNF-α and PAI-2 mRNA in a concentration-dependent manner through reducing the calcium entry by drug-induced depolarization of hypoxic monocytes in an ex vivo model of human endotoxinaemia under hypoxaemic conditions . Koh et al. found that glibenclamide directly reduced the secretion of IL-1β by bone-marrow-derived macrophages in a dose-dependent fashion . In the present study, we further demonstrated that glibenclamide could decrease serum IL-1β and TNF-α level induced by LPS in STZ-induced diabetic mice.
Nalp3 is a ‘general sensor’ for danger signals, representing an important caspase-1-containing inflammasomes and is activated by various pathogens , and damage-associated molecules and environmental irritants . Activation of Nalp3 leads to oligomerization and recruitment of apoptosis-associated speck-like protein and pro-caspase-1, with auto-cleavage and activation of Caspase-1. Active Caspase-1 cleaves pro-IL-1β to active IL-1β, which, when secreted, can exert direct cytotoxic effects as well as recruit other inflammatory cells. In this work, we found that glibenclamide could inhibit Nalp3 inflammasomes and Caspase-1 induced by LPS + high glucose stimulation in cultured peritoneal macrophages, suggesting that the effects of glibenclamide on prevention of serum IL-1β and TNF-α might be related to inhibiting Nalp3 inflammasomes and Caspase-1. The mechanism how glibenclamide inhibits NALP3 inflammasomes and suppresses IL-1β secretion is an intriguing question. Lamkanfi et al. demonstrated that glibenclamide could inhibit the assembly of the NALP3 inflammasomes in response to stimulation with lipopolysaccharide (LPS) and adenosine triphosphate (ATP) . The ability to suppress NALP3 activation of glibenclamide was independent of its inhibitory effect on KATP. The authors speculated that glibenclamide acts the upstream of NALP3 and the downstream of P2X7 . However, the precise molecular target of glibenclamide for its inhibitory effect on NALP3 inflammasome has yet to be identified.